Potential improvements of the cognition of piglets through a synbiotic supplementation from 1 to 28 days via the gut microbiota

The influence of feed supplements on behavior and memory has been recently studied in livestock. The objectives of the study were to evaluate the effects of a synbiotic on: an episodic-like (SOR: Spontaneous Object Recognition), a working (BARR: Fence barrier task), a long-term (TMAZE: Spatial T-maze task) memory test and on gut microbiota composition. Eighteen female piglets were supplemented from 1 to 28 days of age with a synbiotic (SYN), while 17 served as control (CTL). Feces were collected on days 16, 33 and 41 for 16S rRNA gene composition analyses. In the SOR, SYN piglets interacted more quickly with the novel object than CTL piglets. In the BARR, SYN piglets had shorter distances to finish the test in trial 3. In the TMAZE, SYN piglets were quicker to succeed on specific days and tended to try the new rewarded arm earlier during the reversal stage. Difference of microbiota composition between treatments was nonexistent on D16, a tendency on D33 and significant on D41. The synbiotic supplement may confer memory advantages in different cognitive tasks, regardless of the nature of the reward and the memory request. Difference in memory abilities can potentially be explained by differences in microbiota composition.

www.nature.com/scientificreports/ environment, an orange plastic stick (H28 × Ø4 cm) was attached to the back of the farrowing crate in the home pen about 24 h before the novel object recognition test to encourage them to learn to explore a novel object.
The test was carried out during the afternoon between 12:00 h to 20:00 h in the same arena that habituation occurred. The test consisted of two sessions. During the first 10-min session, the piglet was exposed to two similar objects (O1 and O2) attached to each of the side walls in the arena. Then, the piglet was returned to its home pen for a 50-min break. During the second 10-min session, the piglet was exposed to one familiar object (O3) and a completely new one (N1). All the handling of the tested piglet was done by the same familiar experimenter to avoid any supplemental stress. The two different objects used were of the exact same red color and silicone material, with two different shapes: a pasta spoon (L28 × W4 cm) and a spoon rest (L24 × W12 cm). They were attached 14 cm above the floor and 20 cm from the corner of the arena. Objects were washed with soap and water and dried between each trial to minimize odorous cues. The object type and the side of novelty were randomized between piglets. A semi-circular area was designated around the object on both sides (Ø100 cm, with the center matching the middle of the object). The test was videotaped and analyzed by a single trained observer blind to treatment using Ethovision XT 13 14 . The variables analyzed were as follows: the latencies, frequencies and total durations of interactions with both objects (DN1: duration with the novel object; DO3: duration with the old object), position in the designated area next to the novel objects, as well as a discrimination index (DI) = (DN1 − DO3)/(DN1 + DO3) 15 .
Fence barrier task (BARR). A fence barrier task was used to evaluate problem-solving skills and short-term memory at 16 days of age, based on visual clues. This test was adapted from the glass barrier test from Friess et al. 16 . The glass obstacle was substituted by chicken wire fence because of concerns that light reflections from the glass could possibly scare the piglets or impair their visibility of the reward. The feed-motivation reward was substituted by a social-motivation reward using two companion piglets coming from the same litter. It is based on the gregarious instinct of pigs. The unique glass barrier was substituted by two fence barriers in order to make the task more complex. The fences had 25 cm holes on one side (opposite sides for the two fences) to enable the piglet to access the next section of the area.
This test was carried out only during afternoon between 15:00 h to 17:00 h in the habituation arena. The test consisted of a session of five successive trials. To succeed in reaching its siblings, the piglets had to negotiate a path through the two fences by going left and then right (Fig. 1). The piglet had a maximum of 6 min to accomplish the task. Each trial was stopped when the tested piglet touched one of the two companion piglets. At the end of each trial, the piglet was picked up and placed back at the same starting point on the opposite side of the arena from the companion piglets. All the handling of the tested piglet was carried out by the same familiar experimenter to avoid any supplemental stress. The test was videotaped and analyzed by a single trained observer blind to treatment using Ethovision XT 13 14 . The variables analyzed were as follows: total distance travelled, latency to cross the first hole (= front leg crossing) and the overall trial duration. One piglet was removed from the test because of lameness.
Spatial T-maze task (TMAZE). A spatial T-maze task was used to evaluate spatial learning and memory from 33 to 41 days of age.  www.nature.com/scientificreports/ Familiarization with the reward. As this test is based on feed motivation, a 4-day training period was carried out to train the piglets to associate a bowl with a feed reward between 29 to 32 days of age, and also to maintain their comfort with being alone. The reward used was the same chocolate milk used to deliver the synbiotic. Piglets were trained in the habituation arena. The bowl (H9.5 × Ø15 cm) was embedded in a wood box for more stability, and attached to the wall. The habituation to the bowl as container of a feed reward was carried out by a single experimenter with one or two sessions per day, separated at least by a 45-min break while piglets were brought back to their home pen, between 7:00 h to 19:00 h (random assignment to mornings or afternoons). Introduction of the piglets in the arena was always done at the same location of the arena, at the opposite side to the bowl, oriented towards the bowl. When the habituation sessions were carried out, the piglets were no longer being supplemented with the chocolate milk, and this helped maintain their motivation for the milk reward. During the whole process, the amount of milk was progressively reduced. Indeed, bigger volumes were easier to learn to drink in a bowl for the early habituation sessions but for the test it needed to be lowered to avoid satiety. The process followed a progressive pattern: day 1-15 min maximum for a single session, 250 ml of chocolate milk available; day 2-5 min maximum for two sessions, respectively 250 ml and 100 ml available; day 3-5 min then 3 min maximum, respectively 100 ml and 50 ml available; day 4-3 min maximum for two sessions, 30 ml available. All the piglets always managed to succeed before the maximum time allowed. The session stopped when they had drunk the total amount of milk or when they did not show any more interest in it. The variables recorded were the time needed to touch the bowl for the first time as well as the time to drink from the bowl for every trial.
Cognitive test. This test was adapted from Elmore et al. 11 . The T-maze arena was composed of 4 distinct arms (North, South, West and East) of similar dimensions L235 × W70 × H82 cm (see the Supplementary Figure in the Supplementary Information). Only 3 arms were used during each test (West, East, and either North or South). The unused arm was closed by a removable wall. The spatial visual patterns used for the test were made with tape and directly fixed on the walls of the arena (dots, waves, vertical and horizontal strips, respectively for North, South, West and East). The ends of the North and South arms were used as the starting boxes for the trials (two similar areas of L80 × W70 × H82 cm with a sliding fence at the blind ends); whereas the West and East arms, containing a bowl at their blind ends, were used as rewarded and unrewarded arms. The bowl of the rewarded arm was filled with 20 ml of chocolate milk, whereas the bowl of the unrewarded arm was empty and a cup filled of 20 ml of chocolate milk (unreachable by the piglets but providing the same odor cue) was positioned under it in order to prevent piglets from selecting an arm because of milk odor cues. The maze was cleaned between trials, and the experimenter went in both rewarded and unrewarded arms to ensure even spread of human odors and to prevent giving piglets any extra visual cues.
The T-maze test was composed of two distinct stages: a 6-day acquisition stage (A1-6) from 33 to 38 days of age during which the piglet learnt to associate one T-maze arm with the feed reward; followed immediately by a 3-day reversal stage (R1-3) from 39 to 41 days of age during which the rewarded arm was switched compared to the acquisition stage. For both acquisition and reversal stages, piglets did 10 successive trials per day, between 8:00 h to 18:30 h (random assignment to mornings or afternoons). The arm selected to start each trial (5 North and 5 South) was randomized between days, with no more than 2 successive times in the same starting box over the 10 trials pattern. The pattern drawn per day was similar for all the piglets. The objective of these changes was to make sure piglets are not conditioned to always turn left or right but to really use additional cues (visual, odorous) to determine the rewarded arm and to avoid any laterality bias. The rewarded arm (West or East) was also randomized between piglets. Once a piglet found the rewarded bowl, it was given 10 s to drink about 10 ml of chocolate milk. Before the next trial, any feces were cleaned with paper towels, 10 ml of milk was added in the rewarded bowl to refresh it and keep an approximate amount of 20 ml. The milk in the cup under the unrewarded bowl was refreshed between piglets. On A1 and A2 of the acquisition stage, piglets were allowed to go back and forth until they reached the rewarded bowl. The maximum time allowed per trial was 5 min. From A3 to A6 of the acquisition stage and from R1 to R3 of the reversal stage, piglets were no longer allowed to travel back and forth. Four options existed to stop a trial: (1) to select the right arm and drink milk for 10 s, (2) to select the right arm but turn back before reaching the rewarded bowl, (3) to select the wrong arm (piglet allowed to reach the end to check the bowl is empty), and (4) to do not choose any arm for the 60 s after the start of the trial. Video recordings were analyzed using the XP Observer 14 software (Noldus, The Netherlands) by a single trained observer blind to treatment. The variables analyzed were as follows: the proportion of correct choices per day, mean time to make a choice between the two arms (among the 10 trials per day), mean time to touch with its snout the rewarded bowl per day, minimum time to select the rewarded arm per day, number of trials needed to succeed for the first time in the reversal stage and number of trials needed to do two successful trials in the reversal stage. During the reversal stage, if a piglet never succeeded, the maximal 30 trial attempts was attributed.
Six piglets were excluded from this spatial test: 1 for lameness, 3 because of a lack of interest in the reward, 1 because of an error in the trials' pattern on A2, and 1 that found handling to be highly aversive and was too stressed to perform the test. Of the remaining 26 piglets, 6 did not complete the reversal stage because of a too low success rate on A6 (less than 7/10 successful trials per day which can be due to a non-understanding of the task or a lack of motivation for the reward selected).
Microbial analyses of feces. Samples for 16S rRNA gene composition analyses came from fresh feces collected at 16 days of age (during the SOR test) while still with the sow and at 33 and 41 days of age (during the first and last days of the T-maze test). Feces were put on ice and stored at − 80 °C until DNA extractions.
The DNA was extracted from 200 mg of frozen feces by bead beating using the Fast DNA SPIN kit for feces (MP Biomedicals Corporation, Irvine, CA, USA). Extracted DNA was then sent to the Argonne National Sequence processing and microbial community analyses. Briefly, 16S rRNA gene sequences were processed and clustered using the mothur v.1.39.3 standard operating procedure (SOP) designed for MiSeq data 17 ; the mothur MiSeq SOP (version 132) was accessed in August 2018. The SILVA-based bacterial reference alignment was used to identify the taxonomy of OTUs at a cluster cutoff of 97% sequence identity. Measures of richness, evenness and diversity were determined in mothur. Richness can be defined as the number of OTUs observed per sample, evenness represents the uniformity of the distribution of OTUs amongst a community across the multiple observed OTUs, while α-diversity is a concept combining both richness and evenness 18 . Richness and α-diversity were determined through the coverage, the number of OTUs observed, the Chao1, the ACE, the Shannon, the Simpson, the inverse Simpson estimators and β-diversity metrics were calculated using the Yue and Clayton's theta metric (thetaYC, as implemented in mothur) distances 19 . The OTU table was rarefied to a minimum of 1000 sequences per sample to account for differences in sampling effort. A linear model with the treatment (CTL, SYN) and the day of sampling (D16, 33 and 41) was used for the richness, evenness and diversity analyses. The effects of dietary treatments and time periods on the microbial community structure were tested using an analysis of molecular variance (AMOVA) of the thetaYC distance matrix in mothur. Results were adjusted by using the Bonferroni correction. The "metastats" command in mothur was then used to determine the OTUs responsible for the significant differences observed using AMOVA. The "corr.axes" and "otu.association" commands in mothur, specifying the default Pearson method, were then used in combination to estimate the significant Pearson correlations between behavioral indicators and bacterial populations.

Statistical analysis.
Statistical analyses were performed with the software R 3.4.3 20 . The variables of time to drink during the habituation to the bowl of TMAZE, the BARR test variables, as well as the mean times to make a choice and to touch the rewarded bowl were normalized by logarithmic transformation before statistical analysis. Other variables were normal without transformation. In all the statistical analysis, p < 0.05 was considered statistically significant and 0.05 ≤ p < 0.1 as a trend.
Linear model with the treatment as fixed effect was used to test the time for the piglets to touch the bowl the first time during habituation to the TMAZE test, as well as for the variables of the SOR test. For the familiarization phase of the SOR test, the fixed effect was also replaced by the object type and the arena of the test. A mixed effects model for repeated measures with the treatment, the trial or the day and the interaction between the two factors as fixed effects and the animal being included as random effects were used to test the effect of habituation variables to isolation and the TMAZE bowl, as well as the barrier test and the TMAZE test variables. The statistical unit for the previous traits was the animal. These analyses were done with the function lmer from the R package "lme4" 1.1-7. The emmeans function from the R package "emmeans" 1.2-2 was used to perform pairwise comparisons with the FDR correction when interactions were significant (p < 0.05). A generalized linear model (family: Poisson, link: log) was used to analyze the number of trials needed to succeed in the reversal stage of the T-maze, with the treatment as fixed effect and the side of the rewarded arm as a random effect. This analysis was done with the function glmer from the R package "lme4".
Habituation to the arena. There were no interactions between treatment and trial or treatment effects on all habituation variables (p > 0.1). However, there was a trial effect on the distance travelled (p < 0.001) with a significant decrease from the first two trials alone in comparison to the three last ones, on the latency to jump for the first time (p < 0.001) and on the number of jumps (p = 0.039) with a decrease from the first trial alone compared with the other four trials. There was also a tendency toward a decrease in the number of times entering the peripheral area (p = 0.060) from the first trial alone compared with the other four trials, and no effect on the percentage of time spent near the periphery (p > 0.1).
Spontaneous object recognition test. During familiarization, there was no effect of the object used, the test arena or the treatment on the number of interactions (6.5 ± 4.7 times) or the total duration of interactions (25.9 ± 23.8 s) with both objects. There were also no effects on number of times entering the designated area near the objects (7.0 ± 5.6 times) or the total duration of time spent in the designated area (60.3 ± 44.2 s, both p > 0.1).
During the novelty stage, the side of introduction of the new object, as well as the type of object had no effect on all variables recorded (p > 0.1). SYN piglets visited the area with the old object more than CTL piglets (7.8 ± 1.6 times vs. 4.0 ± 0.7 times, p = 0.030). However, their latency to touch the new object was shorter than CTL piglets (see Table 1, p < 0.05). There was no effect of the treatment group on the DI (p > 0.1; Fig. 2 www.nature.com/scientificreports/ Barrier test. The analysis of the variables showed an interaction between treatment and trial for the total distance (p = 0.027) with CTL piglets travelling further than SYN piglets on trial 3 (see Table 1). There was an effect of the trial on all other variables (p < 0.001) with a decrease from trial 1 over the 4 other trials, but no other effects of treatment supplementation on test variables (see Table 1 and Fig. 2 During the test, there were no interactions between treatment and day regarding the number of correct choices per day (p > 0.1). Treatment effect was not significant (p > 0.1) but the day of test was (p < 0.001) with a clear progression within both phases (Fig. 2). On A3, SYN piglets needed less time to touch the rewarded bowl than CTL piglets (13.0 ± 1.9 versus 24.3 ± 5.6 s, p = 0.03).
During the reversal stage, the SYN piglets needed fewer trials to find the reward in the new arm than CTL piglets (10.3 ± 3.3 trials versus 19.8 ± 4.3, p = 0.048).

Figure 2.
Cognitive performances of the pigs of the two treatment diets (CTL: Controls, 5 ml TruMoo ® Chocolate-Whole milk versus SYN: Synbiotics, 3 strains of Lactobacillus at 10 9 CFU /piglet, fructooligosaccharide at 10 mg/day/piglet, beta-glucan at 11 mg/day/piglet, vitamin C at 10 mg/day/piglet diluted in 5 ml of chocolate milk). 1 Letters differ at p < 0.05 between days; *p < 0.05. 2 A: trials during the acquisition phase; R: trials during the reversal phase.
Effects of treatments and day of sampling on microbiota composition. The microbial community structure and its change over time is graphically presented with respect to relative abundances of genera (Fig. 3), β-diversity (Fig. 4) and taxa found to be linear discriminants of CTL and SYN treatments (Fig. 5). The effects of supplementation treatments and time of sampling are presented in Table 3. Across treatments, β-diversity did not reveal significant clustering of overall community structure by treatment, but within each treatment group, the microbiota composition was different over time (p < 0.05). Interestingly, linear discriminant analysis revealed that the succession of gut microbiota over time varied across CTL and SYN treatment. At day 16, CTL and SYN piglets had similar microbiota composition (p > 0.1). However, some taxa were specific to a treatment diet. SYN piglets had Campylobacter, Fusobacteirum, Cloacibacillus, Eubacterium, Fusobacteriaceae and Akkermansia while CTL had not; while CTL had Romboutsia and Alistipes. At day 33, there was a tendency for CTL microbiota composition to be different from SYN composition (p = 0.066). In contrast, SYN piglets had Alloprevotella, Erysipelotrichaceae; while CTL piglets had Holdemanella, Phascolarctobacterium and Fusicatenibacter. At day 41, CTL and SYN piglets had significantly different microbiota composition (p = 0.047) with more Prevotella, Lachnospiraceae and Ruminococcaceae for CTL piglets. There was a tendency for Firmicutes/Bacteroidetes ratio to be higher in CTL group than in SYN group (p = 0.079) and also higher at day 41 in comparison to day 16 (p = 0.062). The treatment had no effect on the percentage of Actinobacteria or Proteobacteria (p > 0.1). However, SYN piglets had a higher percentage of Bacteroidetes (p = 0.017) and a lower percentage of Firmicutes (p = 0.012) than CTL piglets. The presence of Clostridium sensu stricto and Treponema was specified to SYN piglets; while CTL piglets had Streptococcus and SYN piglets had not. The percentage of Actinobacteria did not change over time (p > 0.1), whereas percentage of Bacteroidetes was lower at days 33 and 41 in comparison to day 16 (p = 0.014 and p = 0.011, respectively), the percentage of Firmicutes was also lower at days 33 and 41 in comparison to day 16 (p = 0.0070 and p = 0.0028, respectively) and day 33 was lower than day 41 (p = 0.033). The percentage of Proteobacteria decreased between day 15 to days 33 and 41 (p = 0.020 and p = 0.015, respectively).
Relation between bacteria taxa and behavioral traits. The duration to succeed in trial 2 of the BARR test was the only trait significantly associated with bacterial populations (p = 0.05; Supplementary Table 2

Discussion
In livestock, there is a gap of knowledge about the potential effects of feed additives on general behaviors and even more when looking at effects on learning and memory processes. When using a specific feeding strategy, probiotics, prebiotics or synbiotics, the postulated hypothesis is that the supplementation will affect the gut microbiota composition. Indeed, diet and feed additives are powerful tools to modulate the gut microbiota composition 2,21 . Those alterations of the gut microbiota composition are increasingly understood to affect brain and cognitive functions through the gut-brain axis 2 . Many recent reviews about changes in gut microbiota composition, especially with probiotics and prebiotics, realized in rodents and humans have raised awareness about the impact of feeding on stress 22,23 , anxiety 24 , mood 25 , social behavior 26,27 and cognition 28,29 . Looking especially at cognition, studies in rodents have demonstrated effects on working, spatial and non-spatial memories. In pigs, few studies have confirmed that feeding can impact learning and memory [30][31][32][33] . However, none of the studies in livestock looked at the repercussions of the feed on the gut microbiota composition, as a potential explanation for changes in behaviors and cognitive functions. www.nature.com/scientificreports/ Memory is also sensitive to stress, especially chronic stress 34 . Stress can also change microbiota composition 35 through, for example, modification of gut permeability [36][37][38] . Specific feeding strategies, in pigs, also demonstrated beneficial effect in terms of stress and fearful emotions using a large range of feed supplementation: vitamin E 39,40 , magnesium 41 , tryptophan [42][43][44][45] , aromatic plant extracts 40,46 , chitosan 47 , and the ratios of fat, cholesterol, carbohydrate 48 and linoleic acid in the diet 49 . The use of feed additives can then be beneficial for both stress and memory abilities in pigs, but still remains an untapped solution to ameliorate problem of welfare in swine production.
In the current study, female piglets supplemented with the synbiotic demonstrated some improved aspects of memory in certain tests. They showed some improved working memory performance in a fence barrier task test, were quicker to learn during the acquisition stage of a T-maze solving task and showed higher learning flexibility during the reversal stage of the T-maze. It confirmed previous findings about effects on working and reference memories in pigs with other feeding supplements: high fat or sugar diets 31,33 and dietary sialic acid 30,32 . Both high fat or sugar diets impaired working and reference memories of male Gottingen minipigs 31 . However, exposition of piglets for 8 weeks prior to birth to a high fat and high sugar diet showed improved working and reference memories 33 . In the two studies mentioned, a potential mechanism, proposed by the authors, to explain the diets' effects on memory can be through the cholesterol level. Indeed, cholesterol is involved in synapse formation and synaptic structural plasticity of brain cell membranes, important for the development of memory abilities 50,51 . The brain may require a supply in triglycerides within a range to allow optimal development. Dietary sialic acid supplementation improved the performance of piglets in an 8-arm radial maze 30 and in a T-maze 32 . Sialic acid is involved in neurogenesis 52 , neural repair and learning and memory 53 . The only study about supplementation with a probiotic for livestock was found in poultry. Supplementation of 1-day old Japanese quail for 36 days with the probiotic Pediococcus acidilactici improved their spatial memory in a holeboard-like test 54 .
Pigs in the current study were supplemented from 24-h after birth until 28 days. The early-life period is of high sensitivity for the organism, changes that occurs during that period can change the structure and the development of organs, as the brain 55,56 . Therefore, alterations of gut microbiota in early-life, with a severe stress or supplementation can produce long-term repercussions on the animal development 57 . Piglets from the SYN and CTL tended to differ in gut microbiota composition on day 33 and differed on day 41. Supplementation stopped on day 28, yet gut microbiota remained divergent 5 to 13 days later, suggesting that the synbiotic orally administrated daily may exert influence even after feeding has stopped 57 . The differences of gut microbiota composition between SYN and CTL piglets is a potential explanation to justify the difference of memory abilities between the two treatment groups. Indeed, several pathways of communication between the gut and the brain exist: through the neuronal, endocrine or immune systems 2,58-60 . In rodents, it has been demonstrated that alterations of gut microbiota were correlated with changes in brain-derived neurotropic factor and c-fos proteins concentrations, both known for their implications in memory functions [61][62][63] . In pigs as well, supplementation for a 28-day period with a higher crude protein diet showed an increase in dopamine concentration in the brain stem 64 . Probiotics can modulate the two main actors of gut-brain communication: cytokines 65 and tryptophan, a precursor of www.nature.com/scientificreports/ serotonin, a brain neurotransmitter 66 . It was also showed that microbiota composition changes can modulate levels of brain neurotransmitters, as dopamine and serotonin 67 involved in cognitive functions 68 . SYN treatment did not influence overall diversity of the gut microbiota, suggesting that community richness and evenness were not likely related to SYN mechanisms of influence on behaviour. Interestingly, although differences among CTL and SYN animals were modest when compared between groups at each time point, CTL and SYN microbiomes displayed distinct successional trajectories. Our data suggest that microbiota composition on day 33 tended to be different between the two treatment groups, and these developed to significantly different communities by day 41; however, the paths by which the CTL and SYN microbiomes reach these differences are also distinct. For example, Paraprevotella and Alloprevotella are linear discriminants of SYN but not CTL microbiomes on Day 33, though the mechanistic drivers and functional outcomes of this are unknown. Improvements in terms of memory in the present study were already visible on day 15 while the microbiota composition analysis between the two experimental groups was not significantly different. The most likely explanation for the absence of microbiota distinction on day 15 while cognitive effects were already observed may be due to observation of microbiome structures through 16S DNA analyses of fecal samples. Feces are a mirror of the real gut microbiota composition but still differ from it 69 . The difference induced by the supplementation may have been too subtle to be detectable in the fecal samples but already present in the gut. Moreover, alterations in microbial function in the gut may, through the production or consumption of different metabolites, influence host physiology before the differences in abundances (which require significant growth to become apparent) manifest. Specific bacteria have already been identified with a particular interest for their effects on cognitive functions. In rodents, Mycobacterium vaccae, a commensal bacterium, is as a modulator of cognition, acting both through the immune and serotonergic systems [70][71][72] . In the present study, this specific bacterium did not differ between the two experimental groups. In humans, high levels of Bifidobacteria and Bacteroidetes species were correlated with improvements in working memory 73 . SYN piglets had higher cognitive abilities and a higher percentage of Bacteroidetes in comparison to the CTL piglets. We also demonstrated that the total duration on the second trial of the BARR test was the only cognitive trait significantly correlated with specific bacterial populations, in that case phylum-level categorizations of Bacteroidetes and Firmicutes spp. When looking at general behavioral traits instead of memory traits, the number of significant correlations between traits and bacterial populations is rather high 27 . The lack of correlations between cognitive traits and bacteria may suggest a higher complexity and number of intermediates in the communication between the gut and learning/memory functions. www.nature.com/scientificreports/ The current study also evaluated different types of memories (episodic, working and spatial) on the same individuals at different time points, what makes possible comparisons between different types of memories and within a memory overtime. Pigs spending more time interacting with the new object in the SOR had the worse working memory both in BARR and TMAZE tests. The correlations estimated in the present pigs supported studies done in humans, where it has been demonstrated that similar frontal regions are involved in both working memory and episodic memory processes 74 . Pigs paying more attention to the familiar object in the SOR test also needed more trials to succeed two successive times during the reversal stage of the TMAZE test. The number of trials needed to succeed two successive times during the reversal stage might be associated with a more flexibility in learning abilities. Working memory performances at days 15 during the SOR test and 33 during the TMAZE test were correlated, showing a stability of working memory performance over time.

Conclusion
The supplementation of female piglets from 24-h after birth to 28 days of age with a synbiotic appeared to confer advantages in two of the three distinct cognitive tasks, regardless of the nature of the reward (social or food) and the type of memory requested (working and/or spatial). Oral supplementation in early life for 28 days may result in sustained change in gut microbiota composition. Performance in a specific memory task seemed to be predictive of performance of the individual over time with the same memory or in other tasks requested different type of memory. Table 3. Effects of two different treatment diets (CTL: Controls, 5 ml TruMoo ® Chocolate-Whole milk versus SYN: Synbiotics, 3 strains of Lactobacillus at 10 9 CFU /piglet, fructo-oligosaccharide at 10 mg/day/piglet, beta-glucan at 11 mg/day/piglet, vitamin C at 10 mg/day/piglet diluted in 5 ml of chocolate milk) and day of sampling (D16, D33 and D41) on 16S rRNA gene microbiota composition. Analysis of molecular variance (AMOVA) using the standardized distance matrix method in mothur adjusted by using Bonferroni correction. The determination of the bacteria responsible for the AMOVA significant differences were analyzed with the command "metastats" in mothur from the mothur standard operating procedure (SOP) designed for MiSeq data 17 . The mothur MiSeq SOP was accessed in August 2018. a Bacterial taxa mentioned had a p-value below 0.001.